1. FIELD OF THE INVENTION
The present invention relates to a method and apparatus for the sequential degradation of protein or polypeptides using chemical degradation and solid phase sequencing, and more particularly, this invention relates to a novel method for the microsequencing of very small amounts of protein and identification of the resultant amino acid derivatives.
2. ART BACKGROUND
In 1957, P. Edman published a paper which began a new era in field of protein sequencing, enabling eventually the automated sequencing of proteins and peptides in a liquid phase. P. Edman, Acta. Chem. Scand. 10, 761 (1957).
The three step process first involves coupling the N-terminal amino acid of a starting polypeptide to phenylisothiocyanate (PITC) in the presence of an acid scavenger in an alkaline aqueous or anhydrous solvent. The volatile excess reagent is removed in vacuo (homogeneous phase reaction) or by washing a polypeptide immobilized on an insoluble support (heterogeneous phase reaction) and byproducts of the reaction resulting from the decomposition of PITC are similarly removed, to yield the phenylthiocarbamyl (PTC) polypeptide.
In the second step, the PTC polypeptide is subjected to cleavage by a volatile anhydrous acid to afford the 2-anilino-5(4H)-thiazolone (ATZ) of the N-terminal amino acid and the salt of the residual polypeptide, which is the starting polypeptide with the N-terminal amino acid removed.
The ATZ amino acid is extracted from the residual polypeptide or washed from the insoluble support in the cleavage acid which is subsequently removed in vacuo or evaporated under a stream of nitrogen gas at elevated temperature. In the third step, the ATZ amino acid, is ordinarily subjected to conversion to the more stable phenylthiohydantion (PTH) amino acid. The conversion reaction can be effected thermally or by heating the ATZ amino acid in aqueous, methanolic or anhydrous acid.
The resultant PTH amino acid is identified chromatographically while the shortened residual polypeptide is reacted with PITC to initiate the next cycle of degradation. The foregoing steps (coupling, cleavage and conversion) are repeated for each N-terminal amino acid in the polypeptide.
In 1967 Edman taught in, Edman et al., "A Protein Sequenator" European J. Biochem. 1 (1967) 80-91 a "spinning cup" automated sequencer which permitted the automated sequencing of peptides, still using a liquid phase separation.
In the late 1960's and early 1970's, a modification of the basic Edman chemistry was developed which involved the concepts of Edman degradation as applied using solid phase chemistry. These developments, pioneered by Richard Laursen, Eur. J. Biochem 20 (1971) 89-102 "Solid Phase Edman Degradation-An Automatic Peptide Sequencer" enabled researchers to bind the protein or peptide to be sequenced onto a solid resin, and to pump the Edman reagents past the bound peptide to provide the chemistry that sequentially degraded the peptide.
For background, it is relevant to review the Edman and thioacylation degradation techniques.